Project Overview:The goal of this project is to identify novel small molecule probes that inhibit alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a major new therapeutic target to retard the progression of Parkinson's disease (PD). The 5'UTR of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Probes that can successfully reduce alpha-synuclein expression levels as measured in this luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5'UTR stem-loop to confirm that probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein 5'UTR mRNA stem-loop, which is fused to a luciferase reporter.

Assay Overview:In the ELISA orthogonal screen confirmation of alpha-synuclein tranlation inhibitors was sought. The ELISA used SH-SY5Y neuroglioblastoma cells that were not transfected. Compound was tested in 4-point dose in an effort to observe a dose-response effect.

Dose Response Data Analysis:Three independent wells were evaluated for each compound concentration. Negative control condition (DMSO) was included.Live Cell Correction Factor at dose was calculated as: OD Alamar Blue at dose/ OD Alamar Blue at DMSOELISA values are the average of the independent wells for each compound concentration. The ELISA included a standard curve of recombinant alpha-synuclein (provided in the kit). The ELISA value is background normalized to the ELISA kit using the zero condition as described in the kit manual. ELISA values for each compound concentration are adjusted for live cells as follows:OD ELISA at dose/ Live Cell Correction Factor at doseThe alpha-synuclein concentration values are extrapolated from the recombinant alpha-synuclein standard curve fit equation. Percent inhibition of alpha-synuclein was calculated based on the assumption that complete (100%) inhibition is attainable, as follows:100 - (100 * (alpha-synuclein concentration at dose/ alpha-synuclein concentration at DMSO))